Bone Marrow is the major source of both lymphocytes (immune cells) and erythrocytes in the adult. Among the various cells that constitute the bone marrow are primitive hematopoietic pluripotent stem cells and progenitor cells. An important property of stem cells is their ability to both proliferate, which ensures a continuous supply throughout the lifetime of an individual, and differentiate into the mature cells of the peripheral blood system. When necessary, a pluripotent stem cell can begin to differentiate, and after successive divisions become committed, thus losing the capacity for self-renewal, to a particular line of development. All of the circulating blood cells, including erythrocytes, leukocytes or lymphocytes, granulocytes and platelets originate from various progenitor cells that are themselves derived from precursor stem cells.
The morphologically recognizable and functionally capable cells circulating in the blood include erythrocytes (red blood cells), leukocytes (white blood cells including both B and T cells), non B- and T-lymphocytes, phagocytes, neutrophilic, eosinophilic and basophilic granulocytes, and platelets. These mature cells are derived, on demand, from dividing progenitor cells, such as erythroblasts (for erythrocytes), lymphoid precursors, myeloblasts (for phagocytes including monocytes, macrophages and neutrophils), promyelocytes and myelocytes (for the various granulocytes) and megakaryocytes for the platelets. As stated above, these progenitor cells are themselves derived from precursor stem cells.
A complex network of soluble factors as well as inter- and intra-cellular interactions regulate the proliferation and differentiation of a finite pool of hematopoietic stem cells (HSC). Adult bone marrow consists of a finite number of self-renewing HSCs that replenish the immune system throughout life. Proliferation and differentiation of hematopoietic cells are regulated by hormone-like growth and differentiation factors designated as colony-stimulating factors (CSF) (Metcalf (1989) Nature 339:27-30). CSF can be classified into several factors according to the stage of the hematopoietic cells to be stimulated and the surrounding conditions as follows: granulocyte colony-stimulation factor (G-CSF), granulocyte-macrophage colony-stimulation factor (GM-CSF), macrophage colony-stimulation factor (M-CSF), and interleukin 3 (IL-3). Hematopoiesis is also regulated by inter-cellular and intra-cellular interactions that involve several adhesion molecules.
The stromal cells are a major compartment of the bone marrow microenvironment. These cells exert functional plasticity by producing molecules that belong to classes that include cytokines, neurotrophic factors, neuropeptides and extracellular matrix proteins. Stromal cells provide a niche for HSC at a site close to the endosteal region. At this site, the oxygen level is the lowest in the bone marrow and perhaps, HSC could be protected from oxygen radicals, insults from chemical compounds and from other insults.
Small amounts of certain hematopoietic growth factors account for the differentiation of stem cells into a variety of blood cell progenitors, for the tremendous proliferation of those cells, and for their differentiation into mature blood cells. For instance, G-CSF participates greatly in the differentiation and growth of neutrophilic granulocytes and plays an important role in the regulation of blood levels of neutrophils and the activation of mature neutrophils (Nagata (1990) “Handbook of Experimental Pharmacology”, volume “Peptide Growth Factors and Their Receptors”, eds. Sporn & Roberts, Spring-Verlag, Heidelberg, Vol. 95/1, pp. 699-722; Nicola, et al. (1989) Annu. Rev. Biochem. 58:45-77). It is also reported that G-CSF stimulates the growth of tumor cells such as myeloid leukemia cells (Nicola & Metcalf (1984) Proc. Natl. Acad. Sci. USA 81:3765-3769; Begley et al. (1987) Leukemia 1:1-8). Other growth factors include, erythropoietin (EPO), which is responsible for stimulating the differentiation of erythroblasts into erythrocytes and M-CSF responsible for stimulating the differentiation of myeloblasts and myelocytes into monocytes.
Growth factors are part of a family of chemical messengers known as the cytokines. Cytokines are among the factors that act upon the hematopoietic system to regulate blood cell proliferation and differentiation. Cytokines are also important mediators of the immune response being secreted by both B and T cells, as well as other various lymphocytes. Cytokines encourage cell growth, promote cell activation, direct cellular traffic, act as messengers between cells of the hematopoietic system, and destroy target cells (i.e., cancer cells). Tachykinins are among the various components involved in the modulation and regulation of the hematopoietic system that cytokines play a role in modulating.
The tachykinins are immune and hematopoietic modulators that belong to a family of peptides encoded by a single copy of the evolutionarily conserved preprotachykinin-I (PPT-1) gene (Quinn, et al. (2000) Neuropeptides 34:292-302). PPT-1 is alternatively spliced into four possible transcripts and is ubiquitously expressed. The tachykinins can be released in the bone marrow and other lymphoid organs as neurotransmitters or from the resident bone marrow immune cells (Rameshwar (1997) supra; Ho, et al. (1997) J. Immunol. 159:5654-5660; Maggi (1996) Pharmacol. Res. 33:161-170; 2-6). In the bone marrow, PPT-1 and other hematopoietic growth factors regulate expression of each other through autocrine and paracrine activities. It is believed that various cytokines induce the expression of the PPT-1 gene in bone marrow mesenchymal cells (Rameshwar (1997) supra). The tachykinin family of peptides exerts pleiotropic functions such as neurotransmission and immune/hematopoietic modulation.
PPT-1 peptides exert both stimulatory and inhibitory hematopoietic effects by interacting with different affinities to the G-protein coupled receptors: NK-1, NK-2 and NK-3 (Krause, et al. (1992) J. Invest. Dermatol. 98:2S-7S). NK-1 and NK-2 expression has been reported in bone marrow cells (Rameshwar, et al. (1997) Leuk. Lymphoma 28:1-10), whereas NK-3 has not been detected. NK-1 is induced in bone marrow cells by cytokines and other stimulatory hematopoietic regulators. NK-2 is constitutively expressed in bone marrow cells that are unstimulated or stimulated with suppressive hematopoietic regulators. NK-1 and NK-2 are not co-expressed in bone marrow cells because NK-1 induction by cytokines is correlated with the down regulation of NK-2. NK-1 and NK-2 are co-expressed in breast cancer, however. In bone marrow cells, NK-1 expression requires cell stimulation whereas its expression in neural tissue is constitutive (Rameshwar (1997) supra; Yao, et al. (1999) Mol. Brain. Res. 71:149-158; Abrahams, et al. (1999) J. Neurochem. 73:50-58). It is believed that a particular cytokine discriminates between the expression of NK-1 and NK-2, which directs the type of bone marrow functions: stimulatory vs. inhibitory (Rameshwar, et al. (1997) supra).
PPT-1 is constitutively expressed in several cancers including breast cancer (Singh, et al. (2000) Proc. Natl. Acad. Sci. USA 97:388-393), but its expression requires induction in normal mammary epithelial cells (Rameshwar, et al. (1997) supra; Rameshwar & Gascon (1997) Acta Haematol. 98:59; Qian et al. (2001) J. Immunol. 166:2553). PPT-1 peptides protect cancer cells from radiation damage (Aalto et al. (1998) Peptides 19:231), prevent apoptosis (Reeve et al. (1994) Biochem. Biophy. Res. Comm. 199:1313), enhance breast cancer cell proliferation (Miura, et al. (2000) Blood 96:1733-1739) and could be produced by hypoxia (Fan et al. (1993) Br. J. Pharmacol. 110:43; Qian et al. (2001) J. Immunol. 167:4600). The association between PPT-1 overexpression in cancers that show preference for bone marrow (Gluck (1995) Canadian J. Oncol. 1:58; Malawer & Delaney (1993) In Cancer. Principles and Practice of Oncology. DeVita et al. (eds.) J. B. Lippincott, Philadelphia, p 2225) could provide insights into bone marrow metastasis.
Substance P (SP), the major tachykinin released in the bone marrow, stimulates hematopoiesis through interactions with the neurokinin-1 (NK-1) receptor, which is resident on bone marrow stroma, immune cells and other lymphoid organ cells. Hence, the expression of NK-1 determines the hematopoietic response of the tachykinins. NK-2 inhibits hematopoiesis by interacting with neurokinin-A, another tachykinin encoded for by the PPT-1 gene. The present inventors have discovered that the stimulatory effects mediated by NK-1 can be changed to hematopoietic inhibition in the presence of the amino terminal of SP, a fragment found endogenously in the bone marrow due to enzymatic digestion of SP by endogenous endopeptidases. Further, dysregulated expression of the PPT-1 gene has been associated with different pathologies such as cancer.
Typically, cancer is due to failure of the immune surveillance system in an individual. Even immunocompetent individuals can succumb to aggressive tumors. However, most endocrine cancers (such as cervical, neuroblastoma, breast, prostate), lung and colon cancers have homing preference for the bone marrow, although breast cancer is linked predominantly to bone marrow. Breast cancer metastasis to the bone marrow is a clinical dilemma since the prognosis for the patient is generally poor. Through the functioning presence of different families of growth factors and other molecules, the bone marrow microenvironment is conducive to the survival and transient changes of breast cancer cell function from an aggressive type tumor cell to a more benign-type cell. This reduction in short-term aggression is part of what allow the breast cancer cell to survive and remain undetectable in the bone marrow for prolonged periods.
Despite the emphasis on regular mammograms and self-examination, a breast cancer patient could present with metastasis with cells from the bone marrow to tertiary site for up to ten years after the start of remission. A major reason for breast cancer evasion in the bone marrow is that therapeutic intensity is limited by toxicity to the finite and limited number of hematopoietic stem cells in the bone marrow. It is believed that breast cancer cells are located in the marrow compartment during early phase of cancer and during remission. The cancer cells from the marrow can invade the bone and other distant organs during metastasis. To develop proper drugs to target cancer cells, two areas of cancer entry to the marrow could be targeted: during entry and at “seeding.”
Breast cancer cells have shown increased expression of PPT-1 and its receptor NK-1 as compared to normal mammary epithelial cells. Specific NK-1 antagonists have inhibited breast cancer cell proliferation, suggesting autocrine and/or intercrine stimulation of breast cancer cells by PPT-1 peptides. Thus, PPT-1 and NK-1 are thought to be important in breast cancer development. Further, since PPT-1 peptides are considered hematopoietic modulators, the relationship of PPT-1 peptides and NK-1 receptor with breast cancer may assist in understanding the early integration of breast cancer cells in the bone marrow (Rameshwar, et al. (2000) Proc. Natl. Acad. Sci. USA 97: 388-393).
Under normal circumstances, the bone marrow is able to respond quickly to an increased demand for a particular type of cell. The pluripotential stem cell is capable of creating and reconstituting all the cells that circulate in the blood, including both red and white blood cells and platelets. As stated, progenitor cells that derive from stem cells can replicate and differentiate at an astounding, if not alarming rate. On average, 3-10 billion lymphocyte cells can be generated in an hour. The bone marrow can increase this by ten-fold in response to need. However, in the throes of a diseased state, the bone marrow may not produce enough stem cells, may produce too many stem cells or various ones produced may begin to proliferate uncontrollably. Further complications arise when these stem cells or their associated progenitors are not able to differentiate into the various morphologically recognizable and functionally capable cells circulating in the blood.
Lymphoproliferative syndromes consist of types of diseases known as leukemia and malignant lymphoma, which can further be classified as acute and chronic myeloid or lymphocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. These diseases are characterized by the uncontrollable multiplication or proliferation of leukocytes (primarily the B-cells) and tissue of the lymphatic system, especially lymphocyte cells produced in the bone marrow and lymph nodes.
Lymphocytes (also called leukocytes) are core components of the body's immune system, which is one of the principal mechanisms by which the body attacks and controls cancers. Lymphocytes, or their derivatives, recognize the foreign antigenic nature of cancer cells or of antibodies associated therewith and attack the cancer cells. Upon exposure to a foreign antigen in the human body, lymphocytes naturally proliferate or multiply to combat the antigen.
B and T cells are two broad sub-types of lymphocyte cells, derived from the bone marrow. T cells undergo a process of maturation in the thymus gland. Mature lymphocytes all have a similar appearance. They are small cells with a deeply basophilic nucleus and scanty cytoplasm. B and T cells circulate in the blood and through body tissues. B cells primarily work by secreting soluble substances called antibodies. Each B cell is programmed to make one specific antibody. When a B cell encounters its triggering antigen, it goes through a process wherein it is changed into many large plasma cells. Hence, B cells give rise to plasma cells, which secrete a specific immunoglobulin (antibodies). T cells also respond to antigens. Some of them (CD4+) secrete lymphokines that act on other cells, thus regulating the complex workings of the immune response. Others (CD8+, cytotoxic) directly contact infected cells and are able to cause lysis thereby destroying the infected cells.
Leukemia and other such B-cell malignancies, such as acute and chronic myeloid and lymphocytic leukemia as well as the B-cell subtype of Hodgkins and non-Hodgkin's lymphoma, are examples of lymphoproliferative syndromes that are significant contributors to cancer mortality. In fact, the majority of chronic lymphocytic leukemias are of B-cell lineage (Freedman (1990) Hematol. Oncol. Clin. North Am. 4:405).
Leukemia can be defined as the uncontrolled proliferation of a clone of abnormal hematopoietic cells. Leukemias are further typically characterized as being myelocytic or lymphocytic. Myeloid leukemias affect the descendents of the myeloid lineage, whereas the lymphocytic leukemias involve abnormalities in the lymphoid lineage. Most B cell leukemias and lymphomas are monoclonal, meaning that all of the related tumor cells are derived from one particular aberrant cell.
Generally, leukemia is a neoplastic disease in which white corpuscle maturation is arrested at a primitive stage of cell development. The disease is characterized by an increased number of leukemic blast cells in the bone marrow and by varying degrees of failure to produce normal hematopoietic cells. The condition may be either acute or chronic. Acute myelocytic leukemia (AML) arises from bone marrow hematopoietic stem cells or their progeny. The term “acute myelocytic leukemia” subsumes several subtypes of leukemia, e.g., myeloblastic leukemia, promyelocytic leukemia and myelomonocytic leukemia and is a form of cancer that affects the cells producing myeloid blood cells in the bone marrow. As stated above, myeloid cells are red blood cells, platelets and all white blood cells (which include: neutrophils, monocytes, macrophages, eosinophils and basophils). Primarily, AML involves abnormal white blood cells of the neutrophil type. Production of blood cells is obstructed and immature cells known as “blast cells” accumulate in the bone marrow. These cells are unable to mature and differentiate properly leading to a significant reduction of normal blood cells in the circulation. The accumulation of blast cells in the bone marrow prevents production of other cell types resulting in anemia and low platelet blood counts. Acute lymphocytic leukemia (ALL) arises in lymphoid tissues and ordinarily first manifests its presence in bone marrow. ALL is primarily a form of cancer that affects the lymphocytes and lymphocyte-producing cells in the bone marrow.
Chronic myelogenous leukemia (CML) is characterized by abnormal proliferation of immature granulocytes, for example, neutrophils, eosinophils and basophils, in the blood, bone marrow, the spleen, liver and sometimes in other tissues. A large portion of chronic myelogenous leukemia patients develop a transformation into a pattern indistinguishable from the acute form of the disease.
This change is known as the “blast crises”. Chronic lymphocytic leukemia (CLL) is a form of leukemia in which there is an excess number of mature, but poorly functioning, lymphocytes in the circulating blood. It is to be noted that the rate of production of lymphocytes is not significantly increased and may in fact even be slower than normal. CLL has several phases. In the early phase, it is characterized by the accumulation of small, mature functionally-incompetent malignant B-cells having a lengthened life span. The late stages of CLL are characterized by significant anemia and/or thrombocytopenia.
The two main types of lymphoma are Hodgkin's and non-Hodgkin's lymphoma. Hodgkin's disease is a cancer of the lymphatic system, the network of lymph glands and channels that occurs throughout the body. The defining feature of Hodgkin's disease is the presence of a distinctive abnormal lymphocyte called a Reed-Sternberg cell. There are five. recognized sub-groups of Hodgkin's disease including lymphocyte rich, nodular sclerosing, mixed cellularity, lymphocyte depleted and nodular lymphocyte predominant (which predominantly affects one isolated lymph node). All other types of lymphoma are collectively known as non-Hodgkin's lymphoma. There are thirty sub-types of non-Hodgkin's type lymphoma.
Traditional methods of treating these B-cell malignancies, which include chemotherapy and radiotherapy, have limited utility due to toxic side effects. Short-term side effects of chemotherapy may include significant toxicity, extreme nausea, vomiting, and serious discomfort. The long-term side effects may include diabetes, other forms of B-cell malignancies, other forms of cancer, heart, lung or other organ disease, fatal bleeding during remission induction, and myelodysplasia. The short-term side effects of radiotherapy may include extreme nausea, vomiting, serious discomfort, sterility and infertility. The long-term side effects of radiotherapy may include other forms of B-cell malignancies, cancer, thyroid gland, spleen or other organ failure. These side effects may be moderated by reduced dosages, however, this also increases the risk of remission.
Another traditional method for treating B-cell malignancies includes either bone marrow or stem cell transplantation. However, these procedures are plagued with exorbitant cost and high rates of failure. It is both difficult and costly to locate a sufficient donor and even when one is located, rejection of the transplanted cells often takes place, which in turn can lead to graft versus host disease. Most often, these treatments also include a combination of both chemo and radiotherapies, hence, the concomitant risks involved therein would apply here as well.
There is, therefore, a need for a more non-evasive treatment for lymphoproliferative diseases related to either an increase or decrease in differentiation, as well as uncontrolled proliferation. The present invention meets this need in the art.